The negative factor (nef) gene encoded at the 3' open reading frame of HIV has been shown to be capable of down-regulation of HIV replication. Recent studies have shown that nef acts specifically to inhibit HIV LTR-directed transcription. Introduction of exogenous copies of the nef gene into a patient's cells may suppress HIV gene transcription and viral replication. Retroviral vectors are presently the most efficient method for the transfer of functional genes into large numbers of lymphoid or hematopoietic cells, including primary bone marrow stem cells. Therefore, we will construct recombinant retroviral vectors capable of transducing the HIV nef gene. Transfer and expression of nef by these retroviral vectors will be studied first in human T lymphocyte and monocyte cell lines, and then in primary human peripheral blood lymphocytes, monocytes and hematopoietic stem cells, as models of the cell types involved by HIV infection. Expression of nef will be documented by Northern and Western blotting to identify vectors which efficiently transfer and express the nef gene. We will next examine the effects of nef gene expression on normal cellular functions including growth properties and differentiation, surface antigen expression by immunophenotyping, and cytokine production. The ability of nef expression to inhibit HIV production will be assessed by challenging cells expressing nef with infectious HIV. Standard parameters to quantitate HIV infection will be used such as Southern blotting, HIV RNA measurement by polymerase chain reaction (PCR) and Northern blot, HIV antigen production, syncytia formation, and virus production. In all, these studies will determine the feasibility of using a nef gene therapy approach to suppress HIV infection in human T lymphocytes and monocytic cells.